Nucleic acid quantification products and processes

ABSTRACT

Described herein are products and processes for nucleic acid quantification, which are in part useful for detecting and determining the nucleotide sequence of rare nucleic acids (i.e., low copy number nucleic acids) in a sample. Such products and processes are useful for reducing the dynamic range among different nucleic acid species.

RELATED PATENT APPLICATIONS

This patent application is a continuation application of U.S.application number 13/129,797, entitled NUCLEIC ACID QUANTIFICATIONPRODUCTS AND PROCESSES, naming Charles R. Cantor as applicant andinventor, and designated by attorney docket no. SEQ-6021-US, which is anational stage of international patent application numberPCT/US2009/065280 filed on Nov. 20, 2009, entitled NUCLEIC ACIDQUANTIFICATION PRODUCTS AND PROCESSES, naming Charles R. Cantor asapplicant and inventor, and designated by attorney docket no.SEQ-6021-PC, which claims the benefit of Provisional Patent ApplicationNo. 61/117,474 filed on Nov. 24, 2008, entitled NUCLEIC ACIDQUANTIFICATION PRODUCTS AND PROCESSES, naming Charles R. Cantor as aninventor, and designated by attorney docket no. SEQ-6021-PV. The entirecontent of the foregoing patent applications is incorporated herein byreference, including all text, tables and drawings.

FIELD

The technology in part pertains to products and processes useful forquantification of nucleic acids.

BACKGROUND

Nucleic acid sequencing has become one of the main analytical techniquesof modern molecular biology. The development of reliable methods forsequencing has advanced the understanding of the organization of geneticinformation and has made possible the manipulations of genetic material(i.e., genetic engineering). There are a variety of methods forsequencing nucleic acid molecules. Historically, the most common methodshave been based on chemical (Maxam and Gilbert sequencing) or enzymatic(Sanger dideoxy sequencing and exonuclease-based sequencing) reactionsthat create specific truncated nucleic acid molecules that are thenseparated by electrophoretic techniques in order to determine theirrelative length. More recently, potentially higher throughputtechniques, including pyro-sequencing, nanopore sequencing technology,hybridization-based sequencing methods, and the use of non-radiationbased technologies for visualization of sequencing results, have beendeveloped. It also has been proposed that scanning tunneling microscopycould be used to directly visualize the sequence of a nucleic acidmolecule.

Additionally, a variety of nucleic acid detection techniques, includingpolymerase chain reaction (PCR), ligase chain reaction (LCR), nucleicacid sequence based amplification, strand displacement amplification,amplification with Q replicase, and numerous hybridization techniques,are utilized to detect the presence of nucleic acids of varyingabundance from a variety of sources. Some strategies combine nucleicacid detection techniques with nucleic acid sequencing methods.

SUMMARY

Provided is a method for quantifying amounts of target nucleic acids ofa biological sample, which comprises: (a) preparing a mixture bycontacting (i) a plurality of target nucleic acids of a biologicalsample (targets) with (ii) a known amount of a counterpart nucleic acidfor each of the targets (counterparts), where each counterpart comprises(i) a nucleotide sequence substantially identical to its target, and(ii) a feature that distinguishes each counterpart from its target,under conditions in which the targets hybridize to their counterparts;(b) compressing the dynamic range of the targets in the mixture; (c)determining the amount of each target and/or counterpart; and (d)quantifying the amount of each target by the amount in (c).

Also provided is a method for identifying target nucleic acids of abiological sample, which comprises: (a) preparing a mixture bycontacting (i) a plurality of target nucleic acids of a biologicalsample (targets) with (ii) a known amount of a counterpart nucleic acidfor each of the targets (counterparts), where each counterpart comprises(i) a nucleotide sequence substantially identical to its target, and(ii) a feature that distinguishes each counterpart from its target,under conditions in which the targets hybridize to their counterparts;(b) compressing the dynamic range of the targets in the mixture; and (c)identifying each target and/or counterpart.

Provided also is a method for compressing the dynamic range of targetnucleic acids of a biological sample, which comprises: (a) preparing amixture by contacting (i) a plurality of target nucleic acids of abiological sample (targets) with (ii) a known amount of a counterpartnucleic acid for each of the targets (counterparts), where eachcounterpart comprises (i) a nucleotide sequence substantially identicalto its target, and (ii) a feature that distinguishes each counterpartfrom its target, under conditions in which the targets hybridize totheir counterparts; (b) contacting the mixture with a set of capturenucleic acids, where (i) each capture nucleic acid in the setspecifically hybridizes to a target and counterpart, (ii) each capturenucleic acid in the set hybridizes with substantially the same strengthto the target and counterpart to which it specifically hybridizes; and(iii) the amount of each capture nucleic acid is less than highestamount of a target of the biological sample; whereby the dynamic rangeof the targets is compressed.

In the foregoing methods, the targets and counterparts are amplifiedbefore step (b) in certain embodiments, or the targets and counterpartsare amplified after step (b) in some embodiments. In certainembodiments, the feature in the counterpart is a one-nucleotidesubstitution in the sequence substantially identical to its target. Insome embodiments, the feature in the counterpart is one or moreadditional nucleotides appended to the nucleotide sequence substantiallyidentical to its target. In certain embodiments, the ratio of the amountof each target to the amount of its counterpart is between about 1:10and about 10:1.

In some embodiments, step (b) comprises contacting the mixture with aset of capture agents, where: each agent specifically captures eachtarget and its counterpart, and the amount of each of the capture agentsis within a range that compresses the dynamic range of the targets inthe mixture. In particular embodiments, the array of capture agents ison a solid support. The capture agent interacts with the target and thecounterpart with substantially equal affinity in some embodiments. Incertain embodiments, each capture agent is a capture nucleic acid thatcomprises a polynucleotide sequence complementary to a nucleotidesequence of a target.

The target-capture agent melting temperature (Tm), in some embodiments,differs from the counterpart-capture agent Tm by less than or equal toone degree Celsius. In certain embodiments, the amounts of any twocapture agents of the array differ by less than or equal to 50%.

In some embodiments, the sequence of the target is subsequentlydetermined. The sequence of the target, in certain embodiments, isdetermined by a single-molecule sequencing technique (e.g., analyzingthe target with a nanopore device). In some embodiments, thecounterparts are separated from the targets after step (b). Thecounterparts or targets, in particular embodiments, comprise a capturemoiety that binds to a capture agent.

Certain aspects of the technology are described further in the followingdrawings, detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting embodiment of the technology. To acomposition containing nucleic acid target species is added counterpartnucleic acids, where each counterpart species hybridizes to a particulartarget nucleic acid species. In this particular embodiment, ten units ofeach counterpart species is added to targets present in a dynamic rangefrom one unit to one hundred units. The dynamic range of targets in thesample then is compressed by capturing the target-counterpart mixture toaddresses in an array that contain a capture oligonucleotide thatspecifically hybridizes to a target species or counterpart species. Inthis particular embodiment, each address of the array is capable ofbinding a maximum of ten units of each target or counterpart species,which decreases the dynamic range of target nucleic acids in the sample.The presence and amount of each counterpart species then is determined,and the amount of each target species can be determined in theembodiment.

DETAILED DESCRIPTION

Methods herein can be utilized to determine the abundance (e.g.,relative abundance) of a nucleic acid species in a sample. Methodsherein also can be utilized to determine the nucleotide sequence, and/orpresence, absence or amount, of a nucleic acid species present in asample (e.g., low copy number species). In certain nucleic acid samples,nucleic acid species can be present in a range of copy numbers wherethere can be a most prevalent nucleic acid species and the most rarenucleic acid species. Stated another way, certain nucleic acid speciescan be present in large amounts and some nucleic acid species can bepresent in relatively small amounts in a sample, and the nucleotidesequence, and amount, of the relatively rare nucleic acid species can beascertained using methods provided herein. This range in the amounts ofabundant species and rare species in a sample is referred to herein asthe “dynamic range” of nucleic acid species amounts. In certainembodiments, methods incorporate the use of a distinct counterpartnucleic acid for each target nucleic acid of interest in a sample, andreduce the dynamic range of nucleic acid species in the sample.

Thus, methods herein confer the ability to detect low abundance, rare,or unique nucleic acid sequences in a mixed population of nucleic acidsequences, where some of the sequences are abundant. Methods hereinobviate or lessen the requirement for repeatedly analyzing the samenucleotide sequences, as is sometimes the case when relatively rarenucleic acids are assessed, and thereby reduce the consumption ofreagents, time and other resources.

The ability to detect and identify nucleic acids plays an important partin diagnostic analysis in many fields, including, but not limited to,medical, agriculture, military, and forensic applications. Nucleic aciddetection techniques provided herein can be used as diagnostic tools,disease monitoring tools, and as prophetic tools for determining thepredisposition to certain diseases or conditions, for example. It ispossible to detect the presence or absence of viral or bacterialpathogens, in numerous disease conditions, using nucleic acid detectiontechniques herein, in some embodiments. It is also possible to detectthe presence, absence or amounts of particular genes associated withcertain types of cancer using nucleic acid detection techniques providedherein in certain embodiments. Methods herein also find use inmonitoring bacterial contamination at food processing plants, or insoil, water and crops in agricultural settings, for example. Earlydetection allows for removal of contaminated foods from productionlines, or might even prevent contamination if detection techniques weresensitive enough to identify potential pathogens while still low innumber in the soil or water used for agriculture. Similarly, these samehighly sensitive methods are of use in military or defense applications(i.e., monitoring for potential biological weapons), or forensicapplications (i.e., detecting the presence of non-host nucleic acidswhich identify the presence of a pathogenic cause of death), forexample.

Target Nucleic Acids

The term “target nucleic acids” as used herein refers to one or morenucleic acid(s) to be analyzed, detected, quantified, or sequenced (alsoreferred to as “sample nucleic acid”). Target nucleic acids may containone or more regions of interest. As used herein, the terms “region ofinterest” and “nucleotide sequence of interest” refers to nucleic acidsubsequence or species addressed by processes described herein (e.g.,identification, quantification, analysis). Examples of regions ofinterest include, without limitation, a mutation, a single nucleotidepolymorphism, substitution of one or more contiguous nucleotides,deletion of one or more nucleotides, insertion of one of morenucleotides, a microsatellite, repeat nucleotide region, heterozygousallele, homozygous allele, gene sequence or subsequence, non-codingsequence or subsequence and the like.

Target nucleic acid(s) can be from any source or composition, such asDNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA) and the like),RNA (e.g., message RNA (mRNA), short inhibitory RNA (siRNA), ribosomalRNA (rRNA), tRNA and the like), and/or DNA or RNA analogs (e.g.,containing base analogs, sugar analogs and/or a non-native backbone andthe like). A nucleic acid can be in any form useful for conductingprocesses herein (e.g., linear, circular, supercoiled, single-stranded,double-stranded and the like). A nucleic acid may be, or may be from, aplasmid, phage, autonomously replicating sequence (ARS), centromere,artificial chromosome, chromosome, or other nucleic acid able toreplicate or be replicated in vitro or in a host cell, a cell, a cellnucleus or cytoplasm of a cell in certain embodiments. A target nucleicacid in some embodiments is from a single chromosome (e.g., a nucleicacid sample may be from one chromosome of a sample obtained from adiploid organism). When desired, the target nucleic acid can be altered,as known in the art, such that codons encode for a different amino acidthan is normal, including unconventional or unnatural amino acids(including detectably labeled amino acids).

A target nucleic acid can comprise certain elements that can be selectedaccording to the intended use of the nucleic acid. Any of the followingelements can be included in or excluded from a target nucleic acid. Atarget nucleic acid, for example, may include one or more or all of thefollowing nucleotide elements: one or more promoter elements, one ormore 5′ untranslated regions (5′UTRs), one or more regions into which atarget nucleotide sequence may be inserted (an “insertion element”), oneor more target nucleotide sequences, one or more 3′ untranslated regions(3′UTRs), and a selection element. A target nucleic acid may be providedwith one or more of such elements. In some embodiments, a providedtarget nucleic acid comprises, in operable linkage, a promoter, 5′UTR,optional 3′UTR and insertion element(s) by which a target nucleotidesequence is inserted (i.e., cloned) into the template. In certainembodiments, a provided target nucleic acid comprises, in operablelinkage, a promoter, insertion element(s) and optional 3′UTR, and a 5′UTR/target nucleotide sequence is inserted with an optional 3′UTR. Theelements can be arranged in any order suitable for target proteinproduction, and in some embodiments a target nucleic acid comprises thefollowing elements, operatively linked, in the 5′ to 3′ direction: (1)promoter element, 5′UTR, and insertion element(s); (2) promoter element,5′UTR, and target nucleotide sequence; (3) promoter element, 5′UTR,insertion element(s) and 3′UTR; and (4) promoter element, 5′UTR, targetnucleotide sequence and 3′UTR. The terms “operatively linked” and “inoperable linkage” as used herein refers to two or more nucleic acidelements (promoter, 5′ UTR, 3′ UTR, insertion elements, and the like)linked to each other such that each element performs its intendedfunction, irrespective of the distance between the elements. That is,nucleic acid elements, operatively or functionally linked to each other,may be located adjacent to each other or far apart, and functionallyinteract.

A promoter element can be included in a target nucleic acid. A promoteroften interacts with a RNA polymerase, an enzyme that catalysessynthesis of nucleic acids using a preexisting nucleic acid. When thetemplate is a DNA template, an RNA molecule is transcribed beforeprotein is synthesized. Certain promoters that can be utilized are ofviral origin. Certain promoters are tissue specific and drive expressionof the target sequence only in specific tissues. Such sequences arereadily accessed by the artisan, such as by searching one or more publicor private databases, for example, and the sequences are readily adaptedto target nucleic acids described herein.

A 5′ UTR may comprise one or more endogenous elements and may includeone or more exogenous elements with respect to the target nucleic acidbackbone or target sequence. A 5′ UTR can originate from any suitablenucleic acid, such as genomic DNA, plasmid DNA, RNA or mRNA, forexample, from any suitable organism (e.g., virus, bacterium, yeast,fungi, plant, insect or mammal). The artisan may select appropriateelements for the 5′ UTR based upon the transcription and/or translationsystem being utilized. A 5′ UTR sometimes comprises one or more of thefollowing elements known to the artisan: translational enhancersequence, transcription initiation site, transcription factor bindingsite, translation regulation site, translation initiation site,translation factor binding site, ribosome binding site, replicon,enhancer element, internal ribosome entry site (IRES), and silencerelement.

A 3′ UTR may comprise one or more endogenous elements and may includeone or more exogenous elements with respect to the target nucleic acidbackbone or target sequence. A 3′ UTR may originate from any suitablenucleic acid, such as genomic DNA, plasmid DNA, RNA or mRNA, forexample, from any suitable organism (e.g., a virus, bacterium, yeast,fungi, plant, insect or mammal). The artisan can select appropriateelements for the 3′ UTR based upon the transcription and/or translationsystem being utilized. A 3′ UTR sometimes comprises one or more of thefollowing elements known to the artisan: transcription regulation site,transcription initiation site, transcription termination site,transcription factor binding site, translation regulation site,translation termination site, translation initiation site, translationfactor binding site, ribosome binding site, replicon, enhancer element,silencer element and polyadenosine tail. A 3′ UTR often includes apolyadenosine tail and sometimes does not, and if a polyadenosine tailis present, one or more adenosine moieties may be added or deleted fromit (e.g., about 5, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45 or about 50 adenosine moieties may be addedor subtracted).

Target (or sample) nucleic acid may be derived from one or more sources.A source containing target nucleic acid(s) may contain one or aplurality of target nucleic acids. A plurality of target nucleic acidsas described herein refers to at least two target nucleic acids andincludes nucleic acid sequences that may be identical or different. Thatis, the target nucleic acids may all be representative of the samenucleic acid sequence, or may be representative of two or more differentnucleic acid sequences (e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 1000 or more sequences).

A sample may be collected from an organism, mineral or geological site(e.g., soil, rock, mineral deposit, combat theater), forensic site(e.g., crime scene, contraband or suspected contraband), or apaleontological or archeological site (e.g., fossil, or bone) forexample. A sample may be a “biological sample,” which refers to anymaterial obtained from a living source or formerly-living source, forexample, an animal such as a human or other mammal, a plant, abacterium, a fungus, a protist or a virus. The biological sample can bein any form, including without limitation a solid material such as atissue, cells, a cell pellet, a cell extract, or a biopsy, or abiological fluid such as urine, blood, saliva, amniotic fluid, exudatefrom a region of infection or inflammation, or a mouth wash containingbuccal cells, urine, cerebral spinal fluid and synovial fluid andorgans.

Target nucleic acids may first be isolated from a sample source (e.g.,cells, soil, etc) by methods known in the art. Cell lysis procedures andreagents are commonly known in the art and may generally be performed bychemical, physical, or electrolytic lysis methods. For example, chemicalmethods generally employ lysing agents to disrupt the cells and extractthe nucleic acids from the cells, followed by treatment with chaotropicsalts. Physical methods such as freeze/thaw followed by grinding, theuse of cell presses and the like also may be useful. High salt lysisprocedures are also commonly used. For example, an alkaline lysisprocedure may be utilized. The latter procedure traditionallyincorporates the use of phenol-chloroform solutions, and an alternativephenol-chloroform-free procedure involving three solutions can beutilized. In the latter procedures, solution 1 can contain 15 mM Tris,pH 8.0; 10 mM EDTA and 100 ug/ml Rnase A; solution 2 can contain 0.2NNaOH and 1% SDS; and solution 3 can contain 3M KOAc, pH 5.5. Theseprocedures can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y., 6.3.1-6.3.6 (1989), incorporated herein in itsentirety.

A sample also may be isolated at a different time point as compared toanother sample, where each of the samples are from the same or adifferent source. A target nucleic acid may be from a nucleic acidlibrary, such as a cDNA or RNA library, for example. A target nucleicacid may be a result of nucleic acid purification or isolation and/oramplification of nucleic acid molecules from the sample. Target nucleicacid provided for processes described herein may contain nucleic acidfrom one sample or from two or more samples (e.g., from 1 or more, 2 ormore, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more,9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more,15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 ormore samples).

Target nucleic acid may comprise or consist essentially of any type ofnucleic acid suitable for use with processes of the technology. Targetnucleic acid often is in a form that can hybridize to a capture nucleicacid, for example. As used herein, the term “counterpart nucleic acid”refers to a nucleic acid comprising a nucleotide sequence substantiallyidentical to the target nucleic acid, but contains a feature thatdistinguishes the counterpart from its target. The nucleotide sequencein the counterpart nucleic acid that is identical or substantiallyidentical to a target nucleotide sequence, or portion thereof, allowsthe counterpart nucleic acid to hybridize to a capture nucleic acid withsubstantially the same affinity as a target nucleic acid hybridizes tothe capture nucleic acid.

As used herein, the term “capture nucleic acid” refers to a nucleic acidthat comprises a nucleotide sequence complimentary or substantiallycomplementary to a nucleotide sequence in the counterpart and targetnucleic acids. A capture nucleic acid can be used to capture target andcounterpart nucleic acids for dynamic range compression, in certainembodiments. In some embodiments, a capture nucleic acid can be used toseparate target and counterpart nucleic acid for further sequenceanalysis, such as nucleotide sequencing, or hybridization analysis, forexample. The nucleotide sequence of the capture nucleic acid that iscomplimentary or substantially complementary to a nucleotide sequence inthe target and counterpart nucleic acids may exist adjacent to anucleotide sequence of interest, or may reside within or partiallywithin the nucleotide sequence of interest. Some embodiments provide acapture nucleic acid bound to a solid support. Some embodiments providefor use of a capture nucleic acid in solution (e.g., the capture nucleicacid is not linked to a solid support), and sometimes the capturenucleic acid may be free in solution, interacted with target andcounterpart nucleic acids and then linked to a solid support.

Target nucleic acid may be provided for conducting methods describedherein without processing of the sample(s) containing the nucleic acidin certain embodiments. In some embodiments, target nucleic acid isprovided for conducting methods described herein after processing of thesample(s) containing the nucleic acid. For example, a target nucleicacid may be extracted, isolated, purified and/or amplified from thesample(s). The term “isolated” as used herein refers to nucleic acidremoved from its original environment (e.g., natural environment if itis naturally occurring, or a host cell if expressed exogenously), andthus is altered “by the hand of man” from its original environment. Anisolated nucleic acid generally is provided with fewer non-nucleic acidcomponents (e.g., protein, lipid) than the amount of components presentin a source sample. A composition comprising isolated target nucleicacid can be substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% free of non-nucleic acidcomponents). The term “purified” as used herein refers to target nucleicacid provided that contains fewer nucleic acid species than in thesample source from which the target nucleic acid is derived. Acomposition comprising target nucleic acid may be substantially purified(e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greaterthan 99% free of other nucleic acid species). The term “amplified” asused herein refers to subjecting nucleic acid of a sample to a processthat linearly or exponentially generates amplicon nucleic acids havingthe same or substantially the same nucleotide sequence as the nucleotidesequence of the nucleic acid in the sample, or portion thereof.

Target nucleic acid also may be processed by subjecting nucleic acid toa method that generates nucleic acid fragments, in certain embodiments,before providing target nucleic acid for a process described herein. Insome embodiments, target nucleic acid subjected to fragmentation orcleavage may have a nominal, average or mean length of about 5 to about10,000 base pairs, about 100 to about 1,00 base pairs, about 100 toabout 500 base pairs, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 basepairs. Fragments can be generated by any suitable method known in theart, and the average, mean or nominal length of nucleic acid fragmentscan be controlled by selecting an appropriate fragment-generatingprocedure by the person of ordinary skill. In certain embodiments,target nucleic acid of a relatively shorter length can be utilized toanalyze sequences that contain little sequence variation and/or containrelatively large amounts of known nucleotide sequence information. Insome embodiments, target nucleic acid of a relatively longer length canbe utilized to analyze sequences that contain greater sequence variationand/or contain relatively small amounts of unknown nucleotide sequenceinformation.

Target nucleic acid fragments may contain overlapping nucleotidesequences, and such overlapping sequences can facilitate construction ofa nucleotide sequence of the previously non-fragmented target nucleicacid, or a portion thereof. For example, one fragment may havesubsequences x and y and another fragment may have subsequences y and z,where x, y and z are nucleotide sequences that can be 5 nucleotides inlength or greater. Overlap sequence y can be utilized to facilitateconstruction of the x-y-z nucleotide sequence in nucleic acid from asample. Target nucleic acid may be partially fragmented (e.g., from anincomplete or terminated specific cleavage reaction) or fully fragmentedin certain embodiments.

Target nucleic acid can be fragmented by various methods known to theperson of ordinary skill, which include without limitation, physical,chemical and enzymic processes. Examples of such processes are describedin U.S. Patent Application Publication No. 20050112590 (published on May26, 2005, entitled “Fragmentation-based methods and systems for sequencevariation detection and discovery,” naming Van Den Boom et al.). Certainprocesses can be selected by the person of ordinary skill to generatenon-specifically cleaved fragments or specifically cleaved fragments.Examples of processes that can generate non-specifically cleavedfragment target nucleic acid include, without limitation, contactingtarget nucleic acid with apparatus that expose nucleic acid to shearingforce (e.g., passing nucleic acid through a syringe needle; use of aFrench press); exposing target nucleic acid to irradiation (e.g., gamma,x-ray, UV irradiation; fragment sizes can be controlled by irradiationintensity); boiling nucleic acid in water (e.g., yields about 500 basepair fragments) and exposing nucleic acid to an acid and base hydrolysisprocess.

Target nucleic acid may be specifically cleaved by contacting thenucleic acid with one or more specific cleavage agents. The term“specific cleavage agent” as used herein refers to an agent, sometimes achemical or an enzyme, that can cleave a nucleic acid at one or morespecific sites. Specific cleavage agents often will cleave specificallyaccording to a particular nucleotide sequence at a particular site.

Examples of enzymic specific cleavage agents include without limitationendonucleases (e.g., DNase (e.g., DNase I, II); RNase (e.g., RNase E, F,H, P); Cleavase™ enzyme; Taq DNA polymerase; E. coli DNA polymerase Iand eukaryotic structure-specific endonucleases; murine FEN-1endonucleases; type I, II or III restriction endonucleases such as AccI, Afl III, Alu I, Alw44 I, Apa I, Asn I, Ava I, Ava II, BamH I, Ban II,Bcl I, Bgl I. Bgl II, Bln I, Bsm I, BssH II, BstE II, Cfo I, Cla I, DdeI, Dpn I, Dra I, EcIX I, EcoR I, EcoR I, EcoR II, EcoR V, Hae II, HaeII, Hind II, Hind III, Hpa I, Hpa II, Kpn I, Ksp I, Mlu I, MIuN I, MspI, Nci I, Nco I, Nde I, Nde II, Nhe I, Not I, Nru I, Nsi I, Pst I, PvuI, Pvu II, Rsa I, Sac I, Sal I, Sau3A I, Sca I, ScrF I, Sfi I, Sma I,Spe I, Sph I, Ssp I, Stu I, Sty I, Swa I, Taq I, Xba I, Xho I.);glycosylases (e.g., uracil-DNA glycolsylase (UDG), 3-methyladenine DNAglycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNAglycosylase, FaPy-DNA glycosylase, thymine mismatch-DNA glycosylase,hypoxanthine-DNA glycosylase, 5-Hydroxymethyluracil DNA glycosylase(HmUDG), 5-Hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenineDNA glycosylase); exonucleases (e.g., exonuclease III); ribozymes, andDNAzymes. Target nucleic acid may be treated with a chemical agent, andthe modified nucleic acid may be cleaved. In non-limiting examples,target nucleic acid may be treated with (i) alkylating agents such asmethylnitrosourea that generate several alkylated bases, includingN3-methyladenine and N3-methylguanine, which are recognized and cleavedby alkyl purine DNA-glycosylase; (ii) sodium bisulfite, which causesdeamination of cytosine residues in DNA to form uracil residues that canbe cleaved by uracil N-glycosylase; and (iii) a chemical agent thatconverts guanine to its oxidized form, 8-hydroxyguanine, which can becleaved by formamidopyrimidine DNA N-glycosylase. Examples of chemicalcleavage processes include without limitation alkylation, (e.g.,alkylation of phosphorothioate-modified nucleic acid); cleavage of acidlability of P3′-N5′-phosphoroamidate-containing nucleic acid; and osmiumtetroxide and piperidine treatment of nucleic acid.

As used herein, the term “complementary cleavage reactions” refers tocleavage reactions that are carried out on the same target nucleic acidusing different cleavage reagents or by altering the cleavagespecificity of the same cleavage reagent such that alternate cleavagepatterns of the same target or reference nucleic acid or protein aregenerated. In certain embodiments, target nucleic acid may be treatedwith one or more specific cleavage agents (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more specific cleavage agents) in one or more reaction vessels(e.g., target nucleic acid is treated with each specific cleavage agentin a separate vessel).

Target nucleic acid also may be exposed to a process that modifiescertain nucleotides in the nucleic acid before providing target nucleicacid for a method described herein. A process that selectively modifiesnucleic acid based upon the methylation state of nucleotides therein canbe applied to target nucleic acid. The term “methylation state” as usedherein refers to whether a particular nucleotide in a polynucleotidesequence is methylated or not methylated. Methods for modifying a targetnucleic acid molecule in a manner that reflects the methylation patternof the target nucleic acid molecule are known in the art, as exemplifiedin U.S. Pat. No. 5,786,146 and U.S. patent publications 20030180779 and20030082600. For example, non-methylated cytosine nucleotides in anucleic acid can be converted to uracil by bisulfite treatment, whichdoes not modify methylated cytosine. Non-limiting examples of agentsthat can modify a nucleotide sequence of a nucleic acid includemethylmethane sulfonate, ethylmethane sulfonate, diethylsulfate,nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine), nitrous acid,di-(2-chloroethyl)sulfide, di-(2-chloroethyl)methylamine, 2-aminopurine,t-bromouracil, hydroxylamine, sodium bisulfite, hydrazine, formic acid,sodium nitrite, and 5-methylcytosine DNA glycosylase. In addition,conditions such as high temperature, ultraviolet radiation, x-radiation,can induce changes in the sequence of a nucleic acid molecule. Targetnucleic acid may be provided in any form useful for conducting asequence analysis or manufacture process described herein, such as solidor liquid form, for example. In certain embodiments, target nucleic acidmay be provided in a liquid form optionally comprising one or more othercomponents, including without limitation one or more buffers or saltsselected by the person of ordinary skill.

Counterpart Nucleic Acids

Counterpart nucleic acids are representative of each of the targets ofinterest in a sample population. That is, for each target nucleic acidspecies of interest in a sample population there is a correspondingcounterpart nucleic acid, which is at least in part substantiallyidentical and contains a feature that distinguishes the counterpart fromits target. As described above for target nucleic acids, counterpartnucleic acids may by any type of nucleic acid, naturally occurring orsynthetic, may be from any source or composition, and can be in anyform.

The presence, absence or amount of a counterpart nucleic acid can bedetermined by detecting the presence, absence or amount of the one ormore features that distinguish the counterpart from the target nucleicacid. In some embodiments, a feature that distinguishes a counterpartfrom its target is a substitution of one or more nucleotides relative tothe target, which may be detected by a sequence determination method,for example. In some embodiments a feature that distinguishes acounterpart from its target is the addition or deletion of one or morenucleotides relative to the target. In some embodiments a feature thatdistinguishes a counterpart from its target is the substitution,deletion or addition of nucleotides in the complimentary sequence (e.g.target nucleic acid or capture nucleic acid). In some embodiments afeature that distinguishes a counterpart from its target is thepresence, absence or substitution of nucleotides in sequences adjacentto a complementary sequence (e.g., directly connected or spaced by aspacer sequence in the target or capture nucleic acids).

In some embodiments, counterpart nucleic acids may also include one ormore capture agents. Non-limiting examples of capture agents useful forprocesses described herein include without limitation any member of abinding pair, where one member of the pair is in association with asolid phase and another member of the binding pair is association withthe counterpart nucleic acid. In some embodiments, a target nucleic acidmay comprise a capture agent, and sometimes a counterpart nucleic acidincludes one type of capture agent and a target nucleic acid includesanother type of capture agent (e.g., for capturing to different solidphases). Any suitable binding pair can be utilized to effect anon-covalent interaction, including, but not limited to,antibody/antigen, antibody/antibody, antibody/antibody fragment,antibody/antibody receptor, antibody/protein A or protein G,hapten/anti-hapten, biotin/avidin, biotin/streptavidin, folicacid/folate binding protein, vitamin B12/intrinsic factor, or nucleicacid/complementary nucleic acid (e.g., DNA, RNA, PNA). Any suitablebinding pair can be utilized to effect a covalent linkage, including,but not limited to, a chemical reactive group/complementary chemicalreactive group (e.g., sulfhydryl/maleimide, sulfhydryl/haloacetylderivative, amine/isotriocyanate, amine/succinimidyl ester, andamine/sulfonyl halides). Methods for attaching such binding pairs toreagents and effecting binding are known.

The term “solid support” or “solid phase” as used herein refers to awide variety of materials including solids, semi-solids, gels, films,membranes, meshes, felts, composites, particles, and the like typicallyused by those of skill in the art to sequester molecules. The solidphase can be non-porous or porous. Suitable solid phases include thosedeveloped and/or used as solid phases in solid phase binding assays.See, e.g., chapter 9 of Immunoassay, E. P. Diamandis and T. K.Christopoulos eds., Academic Press: New York, 1996, hereby incorporatedby reference. Examples of suitable solid phases include membranefilters, cellulose-based papers, beads (including polymeric, latex andparamagnetic particles), glass, silicon wafers, microparticles,nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, andmultiple-well plates. See, e.g., Leon et al., Bioorg. Med. Chem. Lett.8: 2997 (1998); Kessler et al., Agnew. Chem. Int. Ed. 40: 165 (2001);Smith et al., J. Comb. Med. 1: 326 (1999); Orain et al., TetrahedronLett. 42: 515 (2001); Papanikos et al., J. Am. Chem. Soc. 123: 2176(2001); Gottschling et al., Bioorg. And Medicinal Chem. Lett. 11: 2997(2001). In some embodiments a solid support may be provided in acollection of solid supports. A solid support collection comprises twoor more different solid support species. The term “solid supportspecies” as used herein refers to a solid support in association withone particular solid phase nucleic acid species or a particularcombination of different solid phase nucleic acid species. In certainembodiments, a solid support collection comprises 2 to 10,000 solidsupport species, 10 to 1,000 solid support species or about 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 unique solid supportspecies. The solid supports (e.g., beads) in the collection of solidsupports may be homogeneous (e.g., all are Wang resin beads) orheterogeneous (e.g., some are Wang resin beads and some are magneticbeads).

A counterpart nucleic acid may be from a nucleic acid library, such as acDNA or RNA library, or may contain sequences representative of thosesequences found in a nucleic acid library, such as a cDNA or RNAlibrary, for example. A target nucleic acid may be a result of nucleicacid purification or isolation and/or amplification of nucleic acidmolecules from a sample. Counterpart nucleic acids provided for sequenceanalysis processes described herein may contain nucleic acid from onesample or from two or more samples (e.g., from 1 or more, 2 or more, 3or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 ormore, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 ormore, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or moresamples).

In some embodiments counterpart nucleic acids are synthetic. Syntheticcounterpart nucleic acids may be made by any process known in the art,which produces nucleic acids useable in the embodiments describedherein. Counterpart nucleic acids may be chemically synthesizedaccording to the solid phase phosphoramidite triester method firstdescribed by Beaucage and Caruthers, Tetrahedron Letts., 22:1859-1862,1981, using an automated synthesizer, as described inNeedham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168, 1984, forexample. Purification of oligonucleotides can be effected by nativeacrylamide gel electrophoresis or by anion-exchange high-performanceliquid chromatography (HPLC), for example, as described in Pearson andRegnier, J. Chrom., 255:137-149, 1983.

Counterpart nucleic acids, naturally occurring or synthetic, may bequantified, for example after synthesis and purification, after PCRamplification, or at any step of a process described herein, using anysuitable method known in the art. For example, measuring the intensityof absorbance of a DNA solution at wavelengths 260 nm and 280 nm is usedas a measure of DNA purity. DNA absorbs ultraviolet (UV) light at 260and 280 nm, and aromatic proteins absorb UV light at 280 nm; a puresample of DNA has the 260/280 ratio at 1.8 and is relatively free fromprotein contamination. A DNA preparation that is contaminated withprotein will have a 260/280 ratio lower than 1.8. Quantitative PCR(Q-PCR) processes are known in the art for determining the amount of aparticular DNA sequence in a sample. Also, DNA can be quantified bycutting with a restriction enzyme, electrophoresing products in anagarose gel, staining with ethidium bromide or a different stain andcomparing the intensity of the DNA with a DNA marker of knownconcentration. Nucleic acid also can be quantified by diphenylamine(DPA) indicators by spectrometric detection at 600 nm and use of astandard curve of known nucleic acid concentrations.

Synthetic counterpart nucleic acids may be designed based upon a targetspecies nucleotide sequence. A portion or all of a counterpart nucleicacid, naturally occurring or synthetic, may be substantially identicalto its representative target nucleic acid. In some embodiments a portionor all of a counterpart nucleic acid, naturally occurring or synthetic,may contain regions that are substantially complementary to capturenucleic acids. As referred to herein, “substantially identical” withrespect to sequences refers to nucleotide sequences sharing a certainamount of sequence identity to each other, counterpart nucleic acids andtarget nucleic acids for example. Included are counterpart, target andcapture nucleotide sequences 55% or more, 56% or more, 57% or more, 58%or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% ormore, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more,69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% ormore, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more,80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% ormore, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more,91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more or 99% or more identical to each other.One test for determining whether two nucleotide sequences aresubstantially identical is to determine the percent of identicalnucleotide sequences shared.

As referred to herein, “substantially complementary” with respect tosequences refers to nucleotide sequences that will hybridize with eachother. The stringency of the hybridization conditions can be altered totolerate varying amounts of sequence mismatch. Included are regions ofcounterpart, target and capture nucleotide sequences 55% or more, 56% ormore, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more,62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% ormore, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more,73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% ormore, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more,84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more or 99% or morecomplementary to each other.

Calculations of sequence identity can be performed as follows. Sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencefor optimal alignment and non-homologous sequences can be disregardedfor comparison purposes). The length of a reference sequence aligned forcomparison purposes is sometimes 30% or more, 40% or more, 50% or more,often 60% or more, and more often 70% or more, 80% or more, 90% or more,or 100% of the length of the reference sequence. The nucleotides atcorresponding positions, respectively, are then compared among the twosequences. When a position in the first sequence is occupied by the samenucleotide as the corresponding position in the second sequence, thenucleotides are deemed to be identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, introduced for optimalalignment of the two sequences. Comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a mathematical algorithm. Percent identity betweentwo nucleotide sequences can be determined using the algorithm of Meyers& Miller, CABIOS 4: 11-17 (1989), which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. Percent identity betweentwo nucleotide sequences can be determined using the GAP program in theGCG software package (available at the World Wide Web URL gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters oftenused is a Blossum 62 scoring matrix with a gap open penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5.

Another manner for determining whether two nucleic acids aresubstantially identical is to assess whether a polynucleotide homologousto one nucleic acid will hybridize to the other nucleic acid understringent conditions. Hybridization, under stringent conditions, alsomay be used to determine whether two nucleic acids are substantiallyidentical to each other. As used herein, the term “stringent conditions”refers to conditions for hybridization and washing. Stringent conditionsare known to those skilled in the art and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. , 6.3.1-6.3.6(1989). Aqueous and non-aqueous methods are described in that referenceand either can be used. An example of stringent hybridization conditionsis hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2× SSC, 0.1% SDS at 50° C.Another example of stringent hybridization conditions are hybridizationin 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed byone or more washes in 0.2× SSC, 0.1% SDS at 55° C. A further example ofstringent hybridization conditions is hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2× SSC, 0.1% SDS at 60° C. Sometimes, stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. Often, stringency conditions are 0.5M sodiumphosphate, 7% SDS at 65° C., followed by one or more washes at 0.2× SSC,1% SDS at 65° C. Stringent hybridization temperatures can also bealtered (i.e. lowered) with the addition of certain organic solvents,formamide for example. Organic solvents, like formamide, reduce thethermal stability of double-stranded polynucleotides, so thathybridization can be performed at lower temperatures, while stillmaintaining stringent conditions and extending the useful life ofnucleic acids that may be heat labile.

Counterpart nucleic acids also may be modified or made as derivatives,variants and analogs of RNA or DNA made from nucleotide analogs, single(sense or antisense) and double-stranded polynucleotides, in someembodiments. It is understood that the term “nucleic acid” does notrefer to or infer a specific length of the polynucleotide chain, thusnucleotides, polynucleotides, and oligonucleotides are also included.Counterpart nucleic acids may comprise or consist essentially of anytype of nucleic acid suitable for use with processes of the technology,such as counterpart nucleic acid that can hybridize to a target nucleicacid, or a capture nucleic acid, for example.

Counterpart nucleic acids can include a detectable label in someembodiments. In some embodiments, a target nucleic acid can include adetectable label, and sometimes a target nucleic acid includes one typeof detectable label and a counterpart nucleic acid includes adistinguishably different detectable label. When desired, the nucleicacid can be modified to include a detectable label using any methodknown to one of skill in the art. The label may be incorporated as partof the synthesis, or added on prior to using the counterpart nucleicacid in any of the processes described herein. Incorporation of labelmay be performed either in liquid phase or on solid phase. In someembodiments the detectable label may be useful for detection of targets.In some embodiments the detectable label may be useful for thequantification of bound or unbound nucleic acids (e.g., hybridized orun-hybridized counterpart). In some embodiments more than one detectablelabel may be used to label counterparts or targets. The use of more thanone detectable label (e.g., different types of labels) may facilitatethe detection or quantification of target and counterpart nucleic acids,bound or in solution. Any detectable label suitable for detection of aninteraction or biological activity in a system can be appropriatelyselected and utilized by the artisan. Examples of detectable labels arefluorescent labels such as fluorescein, rhodamine, and others (e.g.,Anantha, et al., Biochemistry (1998) 37:2709 2714; and Qu & Chaires,Methods Enzymol. (2000) 321:353 369); radioactive isotopes (e.g., 1251,1311, 35S, 31P, 32P, 33P, 14C, 3H, 7Be, 28Mg, 57Co, 65Zn, 67Cu, 68Ge,82Sr, 83Rb, 95Tc, 96Tc, 103Pd, 109Cd, and 127Xe); light scatteringlabels (e.g., U.S. Pat. No. 6,214,560, and commercially available fromGenicon Sciences Corporation, Calif.); chemiluminescent labels andenzyme substrates (e.g., dioxetanes and acridinium esters), enzymic orprotein labels (e.g., green fluorescence protein (GFP) or color variantthereof, luciferase, peroxidase); other chromogenic labels or dyes(e.g., cyanine), and other cofactors or biomolecules such asdigoxigenin, strepdavidin, biotin and the like.

Counterpart nucleic acid also may be exposed to a process that modifiescertain nucleotides in the nucleic acid before providing counterpartnucleic acid for a method described herein. A process that selectivelymodifies nucleic acid based upon the methylation state of nucleotidestherein can be applied to counterpart nucleic acid in certainembodiments.

With reference to FIG. 1, a generalized description of the methods ispresented here, and will be described in more detail below. Counterpartnucleic acid (also referred to as counterpart or counterparts) iscontacted with sample nucleic acid containing target nucleic acids (alsoreferred to as target or targets) of interest, as illustrated in FIG. 1,step 1. The combination of counterpart and sample is contacted withcapture nucleic acid, as shown in FIG. 1, step 2. The capture nucleicacid may be bound to a solid support, as illustrated in FIG. 1, step 2or also may be suspended in solution. The counterparts and targets areallowed to interact with the capture nucleic acid, and subsequently ananalysis is conducted, for example the amounts of each target may bedetermined, as illustrated in FIG. 1, step 3. Optionally, the targetsalso may be further analyzed by sequencing or hybridization studies.

Counterparts and targets (sample) may be contacted using any suitablemethod known to in the art. For example, for small sample sets, theartisan may combine the targets and counterparts manually using a singleor a multichannel pipettor. For larger sets of samples or for highthroughput applications using DNA chips or arrays, the methods describedherein are compatible with robotic devices commonly used to automatehigh throughput DNA analysis. A non-limiting example of an automated orrobotic device used for high throughput analysis, and compatible withthe embodiments described herein, is a device referred to as the OasisLM (produced by Telechem International, Inc. Sunnyvale Calif. 94089).This computer-driven biological workstation can be configured with up tofour separate pipette tip heads with the ability to pipette 1, 8, 96,384 or 1536 samples.

A known or predetermined amount of a counterpart nucleic acid often isintroduced to a system for conducting methods described herein. In someembodiments the amount, in terms of units (e.g., amount (e.g.,weight/weight, weight/volume, grams); concentration) of each counterpartadded to a reaction may be kept constant, as illustrated in FIG. 1. Thenumber of units of each counterpart often is kept constant in a reactionto enable a determination of the relative abundance of a target ofinterest, as well as enabling compression of the dynamic range of thenucleic acid species from a sample (discussed further below). In someembodiments the amount of each counterpart added may be varied (i.e.,the number of units is not kept constant for each target in a reaction).The artisan may gain additional information by performing an analysisusing differing amounts of counterpart nucleic acid. The amount ofcounterpart added for illustrative purposes in FIG. 1 is 10 units foreach target. The sample illustrated in FIG. 1 has 3 nucleic acid specieswith regions of interest, targets A, B, and C. The abundance of the 3targets ranges between about 1 unit and about 100 units, forillustrative purposes. In practice, samples may contain many morenucleic acids of interest, and abundances may vary significantly. Therange in abundance for targets in a sample may be between about 1 unitand about 10 units, about 1 unit and about 50 units, about 1 unit andabout 100 units, about 1 unit and about 500 units, about 1 unit andabout 1,000 units, 1 unit and about 5,000 units, about 1 unit and about10,000 units, about 1 unit and about 50,000 units, about 1 unit andabout 100,000 units, about 1 unit and about 500,000 units, and betweenabout 1 unit and 1,000,000 units. A unit as used herein with referenceto a target is a functional designation and can be set at any actualphysical amount by the artisan. A unit can be designated as a copy of asequence, or 10 copies of a sequence. A unit can be defined as an amountthat contains as little as 1 femtogram (fg) of nucleic acid or as muchas 1 milligram (mg) of nucleic acid, and any amount in between, forexample. More specifically, a unit may contain about 1 fg, about 2 fg,about 5 fg, about 10 fg, about 100 fg, about 500 fg, about 1 nanogram(ng), about 2 ng, about 5 ng, about 10 ng, about 100 ng, about 500 ng,about 1 microgram (μg), about 2 μg, about 5 μg, about 10 μg, about 100μg, about 500 μg, or about 1 mg, and the like. The type of units can beheld constant between nucleic acid species or each nucleic acid speciesmay have its own type of unit.

Non-limiting examples of ratios of target to counterpart and the totalnumber of units of nucleic acid added to each reaction are alsoillustrated in the table in FIG. 1. As illustrated in the table in FIG.1 the ratio of target units to counterpart units is in the range ofabout between 10 to 1 (10:1) and 1 to 10 (1:10), for example. Anyconvenient ratio of target to counterpart may be used, in the range ofbetween about 1:10 and about 10:1 in certain embodiments. That is,ratios of target to counterpart of about 1:10, about 1:9, about 1:8,about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about1:1, about 2:9, about 2:7, about 2:5, about 2:3, about 2:1, about 3:10,about 3:8, about 3:7, about 3:5, about 3:4, about 3:2, about 3:1, about4:9, about 4:7, about 4:5, about 4:3, about 4:1, about 5:9, about 5:8,about 5:7, about 5:6, about 5:4, about 5:3, about 5:2, about 5:1, about6:7, about 6:5, about 6:1, about 7:10, about 7:9, about 7:8, about 7:6,about 7:5, about 7:4, about 7:3, about 7:2, about 7:1, about 8:9, about8:7, about 8:5, about 8:3, about 8:1, about 9:10, about 9:8, about 9:7,about 9:5, about 9:4, about 9:2, about 9:1, about 10:9, about 10:7,about 10:3, and about 10:1, may be used to carry out methods describedherein. Target to counterpart ratios outside the ranges given above mayalso prove useful for quantifying the relative abundance of a targetspecies or for compressing the dynamic range of nucleic acids that areeither extremely rare or extremely abundant in a mixed population.

As described above, and illustrated in FIG. 1, in some embodiments thesame amount of each counterpart species can be added. In someembodiments the amounts of the target species may be determined (e.g.,approximate amount), and an amount of counterpart corresponding to theamount of target can be added. That is, an amount of counterparttailored to the calculated (or approximately determined) amount oftarget can be added at step 1 as illustrated in FIG. 1, and the amountsof counterpart species may differ from one another. In some embodiments,the amount of each capture nucleic acid is less than the highest amountof a target of the biological sample.

In some embodiments targets may be amplified, by PCR for example, priorto contact with counterparts. In some embodiments both targets andcounterparts may be amplified subsequent to being contacted with eachother. In some embodiments targets and counterparts may be amplifiedafter dynamic range compression. That is, targets and counterparts maybe amplified after the targets and counterparts have been contacted withthe capture nucleic acids, present on an array for example, where thecapture nucleic acids are present in amounts that allow dynamic rangecompression.

Dynamic Range Compression

In a mixed nucleic acid sample isolated from a sample source, certainnucleic acid species can be present in large amounts and some nucleicacid species can be present in relatively small amounts, and thenucleotide sequence, and presence, of the relatively rare nucleic acidspecies can be difficult to ascertain. This range in the amounts ofabundant species and rare species in a sample is referred to herein asthe “dynamic range” of nucleic acid species amounts. Dynamic rangecompression as referred to herein is a reduction in the number of copiesof the nucleic acid species with the highest abundance. In certainembodiments, the number of copies of the highest abundance nucleic acidspecies are reduced as compared to the number of copies of the nucleicacid species with a lower or the lowest abundance, while maintaining arepresentative sample of each nucleic acid species of interest in thepopulation in some embodiments. Stated another way, dynamic rangecompression in the latter embodiments lowers the ratio of high abundancenucleic acid species to low abundance species (high abundance: lowabundance) in some embodiments. In certain embodiments, the ratio of thehighest number species to the lowest number species is maintained afterdynamic range compression, but the relative amounts of each species isreduced. Conditions often are selected to maintain at least one copy ofeach nucleic acid species of interest. Dynamic range compression alsomay be used to reduce the ratio of moderately abundant nucleic acidspecies as compared to sequences of low abundance, in certainembodiments. The act of dynamic range compression often results inreduction in the total nucleic acid in the sample used for subsequentanalysis. Approaches that take advantage of dynamic range compressionallow for a reduction in time and costs associated with nucleic acidanalysis and sequencing, as fewer resources are allocated to analyzingand sequencing nucleic acid species that were once abundantlyrepresented.

The amount of compression achieved by use of embodiments describedherein can be tailored to the application at hand. In some embodiments,where the nucleic acid population is known with a degree of certainty,compression of all sequences to about the level of the rarest sequenceallows for rapid analysis of the sequences with reduced or no repeatedanalysis. In some embodiments where the nucleic acid population isunknown, the ratio of highest abundance nucleic acid species may bereduced to a lesser degree, optionally allowing for a determination ofwhether some of a target nucleic acid is present in a sample (e.g.,forensic applications). The degree of dynamic range compression cantherefore be tailored to any range the artisan may require for optimalbalance between time and reagent costs, and task needs.

The degree of dynamic range compression can be expressed in terms of afold reduction of a dynamic range ratio, in some embodiments. A dynamicrange ratio (R_(dr)) may be calculated with (i) number of copies orunits of the highest abundance sequence (H), divided by (ii) the numberof copies of the lower, or lowest, copy number nucleic acid in acomposition (L):

R_(dr)=H/L.

The degree of dynamic range compression can be expressed by multiplyingratio R_(dr) by a multiplier between about 1×10⁻⁹ and 0.999. Therefore,the artisan may capture nearly all (e.g., values as high as about 0.999)or significantly less (e.g., values of about 1×10⁻⁶) of a particulartarget/counterpart mixture. Examples of multipliers include, but are notlimited to, multipliers of about 1×10⁻⁷, about 1×10⁻⁶, about 5×10⁻⁶,about 1×10⁻⁶, about 5×10⁻⁴, about 1×10⁻⁴, about 5×10⁻³, about 1×10⁻³,about 5×10⁻², about 1×10⁻², about 5×10⁻¹, and about 1×10⁻¹.

In some embodiments, the compression factor described above is notequivalent to normalization, as each individual target is not compressedrelative to a particular species. In certain embodiments the compressioncan be equivalent to normalization, where all the species are compressedrelative to a species.

In some embodiments, compression of the dynamic range may beaccomplished using capture nucleic acids linked to a solid phase,including, without limitation, a nucleic acid array or DNA chip. In someembodiments, compression of the dynamic range of nucleic acids may beperformed in solution. Dynamic range compression occurs when thetarget-counterpart mixture, as illustrated in FIG. 1, step 1, iscontacted with capture nucleotides or nucleic acids, as illustrated inFIG. 1, step 2, for example. Capture nucleic acids may interact with asolid support. In some embodiments capture nucleic acids may interactwith a solid support in a reversible manner, allowing the separation oftarget and counterpart for separate analysis, after capture for example.

In some embodiments the capture nucleic acid is present in limitingamounts, in solution or associated with an array, for example. In someembodiments capture nucleic acid is present in saturating amounts, whenin solution or associated with an array, for example. In embodimentswhere dynamic range compression is effected by an array, capture may beto specific addresses on the array that contain a captureoligonucleotide species that specifically hybridizes to a target speciesand counterpart species.

Capture nucleic acid may be present (e.g., in solution or on a solidphase) in saturating amounts, or in non-saturating amounts, relative tothe amount of target nucleic acid and counterpart nucleic acid. Thedegree of saturation can be expressed in terms of a ratio (R_(e)), incertain embodiments, where the amount of capture nucleic acid units(Cap) is divided by the amount of target nucleic acid units andcounterpart nucleic acid units (T+Cpt):

R_(s)=Cap/T+Cpt.

In some embodiments, counterpart nucleic acid is saturating when R_(s)is greater than 1, and often when R_(s) is greater than 10. Thus, insome embodiments, R_(s) can be between 1.001 and about 10 (e.g.,partially saturating conditions; R_(s) is about 2, 3, 4, 5, 6, 7, 8 or9) and can be between about 10 and about 1,000,000 (e.g., R_(s) is about100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10000, 50000, 100000, 500000). In someembodiments, counterpart nucleic acid is non-saturating when R_(s) isless than 1, and often when R_(s) is 0.1 or less. Thus, in someembodiments, R_(s) can be between 0.999 and 10⁻⁶ (e.g., R_(s) is about0.1, 0.05, 0.001, 5×10⁻⁴, 1×10⁻⁴, 5×10⁻⁶, 1×10⁻⁶ and 5×10⁻⁶). Asaturating amount of capture agent sometimes includes amounts of captureagent that allow capture of 75% or more, 76% or more, 77% or more, 78%or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% ormore, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more,89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% ormore, 95% or more, 96% or more, 97% or more, 98% or more or 99% or moreof the target and/or counterpart available for capture. In someembodiments, a non-saturating amount of capture agent may be associatedwith an array. A non-saturating amount of capture agent often gives riseto a compression of the dynamic range, due to capture of only a portionof the targets or complexes by the capture agents.

Dynamic range compression often is based on the effective amount ofcapture nucleic acid. The term “effective amount” as used herein refersto the effective amount of capture nucleic acid to which the target andcounterpart nucleic acids are exposed. The effective amount of capturenucleic acid often is less than the total amount of a target nucleicacid species and its counterpart species, where the target nucleic acidspecies is the highest abundance species in the sample. The effectiveamount of a capture nucleic acid can be modulated according to theamount of time the target and counterpart nucleic acids are exposed tothe capture nucleic acids under hybridization conditions. The effectiveamount of capture nucleic acid can be about the entire amount of thenucleic acid at a particular address on an array, for example, when thetime for hybridization is relatively long (e.g., 24 to 48 hours), insome embodiments. The effective amount of capture nucleic acid can beless than the entire amount of the nucleic acid at a particular addresson an array, for example, when the time for hybridization is relativelyshort (e.g., 1 minute), in some embodiments. Thus, the dynamic range canbe compressed when capture nucleic acid is present in saturating ornon-saturating amounts by selecting the amount of time, and conditions,under which the capture nucleic acids hybridize to the target andcounterpart nucleic acids.

Thus, the hybridization timeframe may be manipulated to optimize dynamicrange compression. In some embodiments, relatively short hybridizationtimes may be used (e.g., about 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40,50, 60 minutes), sometimes where capture nucleic acid is saturating. Insome embodiments hybridization can occur over a longer period of time(e.g. about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100hours, or more), sometimes when capture nucleic acid is non-saturating.Hybridization may occur at a substantially linear rate when theconcentrations of the species to be hybridized are high and do not causea rate-limiting bottleneck. Over time, as the concentration of one orboth of the hybridization partners decreases below a threshold level,hybridization rate slows down and approximates an on/off equilibriumreaction. Taking advantage of hybridization rates, it is possible toadjust length of time of hybridization to selectively eliminate highlyabundant species, and a time course can be readily performed to optimizehybridization times.

Capture nucleic acids are configured to interact with both target andcounterpart, and in some embodiments the sequences of the counterpartand target species that hybridize to the capture nucleic acid areidentical. The use of identical sequences gives rise to substantiallyequal interaction affinity with the capture oligonucleotide. The term“substantially equal affinity” as used herein with respect to binding ofdistinct nucleotide species to a common capture nucleic acid refers tobinding reactions and conditions in which each target and counterpartinteracts with a capture nucleic acid, with substantially the samefrequency. In a particular embodiment illustrated in FIG. 1, step 2,each address of the array is capable of binding a maximum of ten unitsof each target-counterpart species, which decreases the dynamic range oftarget nucleic acids in the sample. The presence and amount of eachcounterpart species then is determined, and the amount of each targetspecies can be determined.

Specific hybridization of capture oligonucleotides to a specific targetspecies can be optimized, by hybridization conditions for example,according to the percent identity (% identity) of the capture and targetnucleotide sequences, that hybridize to one another. As referred toherein, percent (%) identity is a measure of the number of identicalbases in two or more nucleotide sequences when the sequences areoptimally aligned and compared. Methods of determining sequence identityare described. Specific hybridization of sequences due to sufficientpercent (%) sequence identity of capture and target nucleic acidssometimes include nucleotide sequences which have 55% or more, 56% ormore, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more,62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% ormore, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more,73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% ormore,79% or more, 80% or more, 81% or more, 82% or more, 83% or more,84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more or 99% or moresequence identity, so long as each capture nucleic acid in the sethybridizes with substantially the same strength, affinity, orhybridization efficiency, to the target and counterpart to which itspecifically hybridizes. The strength of hybridization is due tosequences available for hybridization as well as conditions in whichhybridization is performed. Optimal hybridization conditions can bedependent on length and sequence of the nucleic acids of interest, andcan be readily selected (e.g., certain hybridization conditions aredescribed herein).

In some embodiments dynamic range compression may be performed, at leastpartially or completely, in solution. After target and counterpartnucleic acids are contacted, a limiting effective amount of capturenucleic acid linked to a binding partner may be added to the mixture, insome embodiments. After hybridization, capture nucleic acid hybridizedto target and counterpart nucleic acids can be contacted with a solidphase to which the other member of the binding partner is linked.

In some embodiments, a target nucleic acid includes a region of interest(discussed above). The nucleotide sequence of capture nucleic acids thathybridize to the target nucleic acid can be adjacent to a terminus ofthe region of interest, in some embodiments, and can comprise the regionor interest, or a portion thereof, in certain embodiments. The term“adjacent” as used herein refers to a distance between the termini oftwo subsequences of 0 nucleotides. The term “adjacent” and“substantially adjacent” as used herein can refer to a distance of 1, 2,3, 4, 5, 6, 7, 8, 9, 10 nucleotides between the termini of twosubsequences.

After interaction of capture nucleic acid with a target-counterpartmixture, the nucleic acids may be treated with an agent that removesnon-hybridized nucleic acid, in some embodiments. In certainembodiments, an exonuclease is utilized, which can digest molecules ofnucleic acid not hybridized to the capture nucleic acids. In certainembodiments the target-capture or counterpart-capture nucleic acidcomplexes may be isolated, by solid phase capture, for example, if suchcomplexes have not already been captured (e.g., by direct interactionwith a solid phase array).

Hybridization conditions, including without limitation the meltingtemperature (Tm) of the target-capture complex and counterpart-capturecomplex, are considerations for optimizing dynamic range compression.Additionally, design of capture agents with the ability to distinguishbetween closely related species, by manipulating hybridizationconditions and temperatures, gives the artisan significant power andflexibility for dynamic range compression, and sequence capture,identification and analysis. In some embodiments, the target-captureagent Tm differs from the counterpart-capture agent Tm by less than orequal to one degree Celsius. Melting point temperature differencesbetween target-capture agent and counterpart-capture agent complexesuseful for distinguishing between target and counterpart speciessometimes include differences of 1 degree Celsius or less, 0.9 degreeCelsius or less, 0.8 degree Celsius or less, 0.7 degree Celsius or less,0.6 degree Celsius or less, 0.5 degree Celsius or less, 0.4 degreeCelsius or less, 0.3 degree Celsius or less, 0.2 degree Celsius or less,or 0.1 degree Celsius or less. This difference in hybridizationefficiency allows selective binding and subsequent capture or dynamicrange compression of particular nucleic acid species based on the Tm ofthe target-capture agent and counterpart-capture agent complexes.

With reference to FIG. 1, for embodiments involving capture of targetand counterpart to an array, the capture agents may interact with asolid phase at discrete locations, as opposed to interacting withcapture agents across the entire surface of the solid support. An arrayprepared with capture agents associated with specific discretelocations, of an array, is illustrated in FIG. 1, step 2. Capture agentsinteracting with specific, discrete, locations can be referred to ashaving specific addresses, and the address of each location maybedefined by a row and column location. Preparing solid phase in thismanner allows the artisan to perform a number of different rangecompressions or target and/or counterpart captures on the same array,while still allowing identification of each individual address so thatparameters associated with a particular address can be preserved anddetermined.

In certain embodiments, the amounts of any two capture nucleic acids ina system, with substantially the same affinity for both target andcounterpart nucleic acid, may differ. In certain embodiments, theamounts of capture nucleic acid in an array differ by 50% or less, 49%or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% orless, 43% or less, 42% or less, 41% or less, 40% or less, 39% or less,38% or less, 37% or less, 36% or less, 35% or less, 34% or less, 33% orless, 32% or less, 31% or less, 30% or less, 29% or less, 28% or less,27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% orless, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less,16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% orless, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% orless, 4% or less, 3% or less, 2% or less, or 1% or less.

Subsequent to target and counterpart capture, counterpart nucleic acidmay be separated from target at each address in certain embodiments, andonly the target or the counterpart is further processed, for detectionand/or sequencing for example. In some embodiments the target orcounterpart can be captured by a capture agent partner linked to solidphase via a capture agent on target or counterpart. Advantages of thisapproach are fewer sequencing or detection events are needed to identifyand analyze low abundance nucleic acid targets, which saves time andresources due to fewer sequencing reactions being wasted on highlyabundant sequences.

The length of nucleic acid sequences may affect formation oftarget/capture complex and counterpart/capture complex. Nucleic acidsisolated from samples and containing regions of interest may containsequences of varying lengths, dependent on natural sequence length andnucleic acid breakage/cleavage during isolation and preparation. Often,increasing the length of complementary nucleotide sequences increasesspecificity of hybridization. In some embodiments, each target sequenceof interest may also have sub-regions that are unique or better suitedfor hybridization. The length of target nucleic acid can be selectedbased on sequence composition and conditions. Target nucleic acid lengthcan be about 5 base pairs (bp), 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp,700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp,6000 bp, 7000 bp, 8000 bp, 9000 bp, or 10,000 bp in length, in certainembodiments.

Similarly, the length of counterpart nucleic acid can be selected basedon sequence composition and conditions. Counterpart nucleic acid lengthscan be about 5 base pairs (bp), 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp,700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp,6000 bp, 7000 bp, 8000 bp, 9000 bp, or 10,000 bp in length, in certainembodiments.

Capture nucleic acids are of a sufficient length to include a nucleotidesequence complementary to target nucleic acid and counterpart nucleicacid and allow solid phase capture of capture/target andcapture/counterpart complexes, in certain embodiments. Capture nucleicacid lengths can be about 5 base pairs (bp), 10 bp, 20 bp, 30 bp, 40 bp,50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp,5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, or 10,000 bp in length, incertain embodiments. Capture/target and capture/counterpart complexesmay include no overlapping region, and in some embodiments, may includeone or two overlapping regions. Overlapping regions can be about 5 basepairs (bp), 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 6000 bp, 7000 bp, 8000bp, 9000 bp, or 10,000 bp in length, in certain embodiments. Regions ofoverlap may be significantly overlapping (e.g. greater than 95 or even99% overlap) to partially overlapping (overlap of between about 10% and90%), to minimally overlapping.

Detection and Quantification of Target Nucleic Acid Species

After target and counterpart have been captured, target nucleic acid andcounterpart nucleic acid may be subjected to further analysis, includingwithout limitation, detection, sequencing, hybridization andquantification. In some embodiments target nucleic acid and/orcounterpart nucleic acid species can be detected using a labelincorporated directly onto or into the target or combined with thetarget by way of a hybridized capture agent.

In some embodiments target nucleic acid may be separated fromcounterpart nucleic acid prior to further analysis. The amount ofcounterpart nucleic acid can be determined with fewer reagents, and at alower cost, where there is a relatively large amount of target nucleicacid and a relatively small amount of counterpart nucleic acidhybridized to an array location, in certain embodiments.

Any detectable label suitable for detection of an interaction orbiological activity in a system can be appropriately selected andutilized by the artisan (e.g. certain detectable labels are describedherein). In some embodiments, a known amount of label is linked to atarget nucleic acid or counterpart nucleic acid (e.g., sometimes thelabel is stoichiometric). An amount of detectable label linked to acounterpart nucleic acid can be determined at a location on an array,for example, and an amount of the counterpart nucleic acid can bedetermined based on the amount of label detected.

In some embodiments target nucleic acid species can be further analyzedby nucleotide sequencing. Any suitable sequencing method can beutilized. In some embodiments, nucleotide sequencing may be by singlenucleotide sequencing methods and processes. Single nucleotidesequencing methods involve contacting sample nucleic acid and solidsupport under conditions in which a single molecule of sample nucleicacid hybridizes to a single molecule of a solid support. Such conditionscan include providing the solid support molecules and a single moleculeof sample nucleic acid in a “microreactor.” Such conditions also caninclude providing a mixture in which the sample nucleic acid moleculecan hybridize to solid phase nucleic acid on the solid support. Singlenucleotide sequencing methods useful in the embodiments described hereinare described in International PCT Patent Application NumberPCT/US2009/031169 filed Jan. 15, 2009, published as publication no. WO2009/091934 on Jul. 23, 2009, and incorporated herein by reference, inits entirety.

Examples of sequencing platforms include, without limitation, the 454platform (Roche) (Margulies, M. et al. 2005 Nature 437, 376-380),Illumina Genomic Analyzer (or Solexa platform) or SOLID System (AppliedBiosystems) or the Helicos True Single Molecule DNA sequencingtechnology (Harris T D et al. 2008 Science, 320, 106-109), the singlemolecule, real-time (SMRTTM) technology of Pacific Biosciences, andnanopore sequencing (Soni G V and Meller A. 2007 Clin Chem 53:1996-2001). Such platforms allow sequencing of many nucleic acidmolecules isolated from a specimen at high orders of multiplexing in aparallel manner (Dear Brief Funct Genomic Proteomic 2003; 1: 397-416).Each of these platforms allow sequencing of clonally expanded ornon-amplified single molecules of nucleic acid fragments.

In some embodiments, target nucleic acid species can be analyzed by asingle nucleotide sequencing technology known as pyro-sequencing.Pyro-sequencing is a method of DNA sequencing by synthesis. Sequencingby synthesis involves taking a single strand of DNA and synthesizing thecomplimentary strand enzymatically in a reaction, which is coupled to achemiluminescent enzyme. Successful incorporation of a base liberates apyrophosphate (PPi), which is converted into ATP, which then producesvisible light through a reaction with luciferin. A camera detects theproduction of the visible light. The amount of light liberated isproportional to the amount of ATP produced. A number of pyro-sequencingmethods and devices are available to the artisan, including, by way ofnon-limiting example, the Genome Sequencer FLX with GS FLX Titaniumseries reagents by 454 Life Sciences, a Roche company (Branford, Conn.).

In some embodiments, single nucleotide sequencing may be by the use of ananopore device. Advances in nucleic acid analysis technology haveincluded the use of nanopore technology to determine, for example, thesequence of a nucleic acid. A nanopore is a hole on the order of 1nanometer in internal diameter in either a piece of silicon or naturallyoccurring as a transmembrane protein. When a nanopore is immersed in aconducting fluid and a voltage is applied, an electric current due toconduction of ions through the nanopore is observed. The amount ofcurrent is sensitive to the size of the nanopore. As DNA molecules passthrough a nanopore, the DNA causes a partial blockage that may changethe magnitude of the current, which passes through the nanopore.Detection of which nucleotide is flowing through the pore at any givenmoment is also possible due to the differences in the dimensions of eachnucleotide, as the DNA is passed through the nanopore. The change in thecurrent through the nanopore as the DNA molecule passes through thenanopore represents a direct reading of the DNA sequence. One suchmethod of single nucleotide sequencing using a nanopore device isdescribed in International PCT Patent Application NumberPCT/US2009/031169 filed Jan. 15, 2009, published with publication no. WO2009/091934 on Jul. 23, 2009, and incorporated herein by reference, inits entirety.

The amount of a particular target nucleic acid is quantified in certainembodiments. In some embodiments the amount of target nucleic acidcaptured can be determined from the amount of a counterpart added to asample. When a known amount of a particular counterpart species is mixedwith target, the amount of target can be calculated from the amount ofcounterpart detected after dynamic range compression. For example,counterpart can be mixed with target, the mixture can be contacted withan array having an address populated with a capture nucleic acid thatspecifically hybridizes to the counterpart species and its targetspecies, and the amount of counterpart species hybridized at the addresscan be determined (e.g., by a single molecule sequencing technique). Insome embodiments, the amounts of target species and counterpart speciesare determined and a ratio of the two is calculated. The amount of thetarget species can be inferred, extrapolated or determined by the amountof counterpart species or the ratio (counterpart species to targetspecies or target species to counterpart species) in certainembodiments.

Examples of Embodiments of the Technology

Provided hereafter are non-limiting examples of embodiments of thetechnology.

1A. A method for quantifying amounts of target nucleic acids of abiological sample, which comprises:

-   -   a. preparing a mixture by contacting (i) a plurality of target        nucleic acids of a biological sample (targets) with (ii) a known        amount of a counterpart nucleic acid for each of the targets        (counterparts),    -   wherein each counterpart comprises (i) a nucleotide sequence        substantially identical to its target, and (ii) a feature that        distinguishes each counterpart from its target, under conditions        in which the targets hybridize to their counterparts;    -   b. compressing the dynamic range of the targets in the mixture;    -   c. determining the amount of each target and counterpart; and    -   d. quantifying the amount of each target by the amount in (c).

1B. A method for identifying target nucleic acids of a biologicalsample, which comprises:

-   -   a. preparing a mixture by contacting (i) a plurality of target        nucleic acids of a biological sample (targets) with (ii) a known        amount of a counterpart nucleic acid for each of the targets        (counterparts),    -   wherein each counterpart comprises (i) a nucleotide sequence        substantially identical to its target, and (ii) a feature that        distinguishes each counterpart from its target, under conditions        in which the targets hybridize to their counterparts;    -   b. compressing the dynamic range of the targets in the mixture;        and    -   c. identifying each target and counterpart.

1C. A method for compressing the dynamic range of target nucleic acidsof a biological sample, which comprises:

-   -   a. preparing a mixture by contacting (i) a plurality of target        nucleic acids of a biological sample (targets) with (ii) a known        amount of a counterpart nucleic acid for each of the targets        (counterparts),    -   wherein each counterpart comprises (i) a nucleotide sequence        substantially identical to its target, and (ii) a feature that        distinguishes each counterpart from its target, under conditions        in which the targets hybridize to their counterparts;    -   b. contacting the mixture with a set of capture nucleic acids,        wherein (i) each capture nucleic acid in the set specifically        hybridizes to a target and counterpart, (ii) each capture        nucleic acid in the set hybridizes with substantially the same        strength to the target and counterpart to which it specifically        hybridizes; and (iii) the amount of each capture nucleic acid is        less than highest amount of a target of the biological sample;        whereby the dynamic range of the targets is compressed.

2. The method of any one of embodiments 1A, 1B and 1C, wherein thetargets and counterparts are amplified before (b).

3. The method of any one of embodiments 1A, 1B and 1C, wherein thetargets and counterparts are amplified after (b).

4. The method of any one of the preceding embodiments, wherein thefeature in the counterpart is a one-nucleotide substitution in thesequence substantially identical to its target.

5. The method of any one of the preceding embodiments, wherein thefeature in the counterpart is one or more additional nucleotidesappended to the nucleotide sequence substantially identical to itstarget.

6. The method of any one of the preceding embodiments, wherein the ratioof the amount of each target to the amount of its counterpart is betweenabout 1:10 and about 10:1.

7. The method of embodiment 1A or embodiment 1B, wherein (b) comprisescontacting the mixture with a set of capture agents, wherein:

each agent specifically captures each target and its counterpart, andthe amount of each of the capture agents is within a range thatcompresses the dynamic range of the targets in the mixture.

8. The method of embodiment 7, wherein the array of capture agents is ona solid support.

9. The method of embodiment 7 or 8, wherein the capture agent interactswith the target and the counterpart with substantially equal affinity.

10. The method of any one of embodiments 7, 8 or 9, wherein each captureagent is a capture nucleic acid that comprises a polynucleotide sequencecomplementary to a nucleotide sequence of a target.

11. The method of embodiment 10, wherein the target-capture agentmelting temperature (Tm) differs from the counterpart-capture agent Tmby less than or equal to one degree Celsius.

12. The method of any one of embodiments 7-10, wherein the amounts ofany two capture agents of the array differ by less than or equal to 50%.

13. The method of embodiment 1B or 1C, wherein the sequence of thetarget is subsequently determined.

14. The method of embodiment 13, wherein the sequence of the target isdetermined by analyzing the target with a nanopore device.

15. The method of embodiment 1A or 1B, wherein the counterparts areseparated from the targets after (b). 16. The method of embodiment 15,wherein the counterparts or targets comprise a capture moiety that bindsto a capture agent.

17. The method of embodiment 1C, which comprises separating thecounterparts from the targets after (b).

18. The method of embodiment 17, wherein the counterparts or targetscomprise a capture moiety that binds to a capture agent.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” is about 1, about2 and about 3). For example, a weight of “about 100 grams” can includeweights between 90 grams and 110 grams. Thus, it should be understoodthat although the present technology has been specifically disclosed byrepresentative embodiments and optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and such modifications and variations are consideredwithin the scope of this technology.

Embodiments of the technology are set forth in the claims that follow.

1-21. (canceled)
 22. A method for quantifying the amount of a targetnucleic acid of a biological sample, which comprises: (a) contacting (i)a target nucleic acid of a biological sample with (ii) a known amount ofa counterpart nucleic acid for the target, where the counterpartcomprises (i) a nucleotide sequence substantially identical to itstarget nucleic acid, and (ii) a feature that distinguishes thecounterpart nucleic acid from the target nucleic acid, under conditionsin which the target hybridizes to the counterpart nucleic acid; (b)amplifying the target and the counterpart nucleic acid to form a mixturecomprising the amplified target nucleic acid and the amplifiedcounterpart nucleic acid; (c) determining the amount of the amplifiedtarget nucleic acid and the amplified counterpart nucleic acid; and (d)quantifying the amount of the target nucleic acid based on the ratio ofthe amount of the amplified target nucleic acid and the amount of theamplified counterpart nucleic acid.
 23. The method of claim 22, whereinthe target nucleic acid is contacted with differing predeterminedamounts of the counterpart nucleic acid.
 24. The method of claim 22,wherein the feature in the counterpart is a one nucleotide substitutionin the sequence substantially identical to its target.
 25. The methodsof claim 22, wherein the method step (b) further comprises contactingthe mixture with a capture nucleic acid, and wherein the capture agentis a capture nucleic acid that comprises a polynucleotide sequencecomplementary to a nucleotide sequence of a target.
 26. The methods ofclaim 25, wherein the capture agent is on a solid support.
 27. Themethods of claim 25, wherein the capture agent interacts with the targetand the counterpart with substantially equal affinity.
 28. The method ofclaim 25, wherein the capture agent melting temperature differs from thecounterpart-capture agent melting temperature by less than or equal toone degree Celsius.
 29. The method of claim 25, wherein the counterpartnucleic acid or target nucleic acid comprises a capture moiety thatbinds to the capture agent.
 30. The method of claim 22, wherein theratio of the amount of target nucleic acid to the amount of itscounterpart nucleic acid is between 1:10 and 10:1.
 31. A system forquantifying the amount of a target nucleic acid of a biological sample,wherein the system comprises: (a) a target nucleic acid of thebiological sample, (b) a known amount of a counterpart nucleic acid forthe target nucleic acid, (c) a device configured for (i) amplifying thetarget nucleic acid, (ii) quantifying the amplified target nucleic acidand the counterpart nucleic acid, and (iii) determining the ratio of theamount of the amplified target nucleic acid and the counterpart nucleicacid.